This invention relates generally to tissue microarray technology. Microarray technology allows the rapid analysis of hundreds to thousands of genes, mRNAs, proteins, and tissue samples in expedited experimental approaches and is used to identify and characterize genes and markers involved in a variety of human pathologies.
Recently developed high density tissue microarray technology involves arraying up to thousands of cylindrical tissue cores from individual tumors on a tissue microarray (see, e.g. Kononen et al. Nat Med. 1998 July ;4(7):844-7). More than two hundred serial sections can then be made from an individual microarray block and used for analysis of DNA, RNA, and/or proteins on a single glass slide. The technology is useful in that it allows rapid analysis of a large number of samples so that the statistical relevance of new markers can be determined in a single experiment. In addition, altered expression levels can be correlated to amplification or deletion events in specific tumor samples using serial sections, allowing simultaneous determination of gene copy number and expression analysis of candidate pathogenic genes and suppressor genes. Arrays have been made containing numerous tumor types (see, e.g. Schraml et al. Clin Cancer Res. 1999 August ;5(8):1966-75) as well as multiple stages and grades within individual tumor types (see, e.g. Moch et al. Am J Pathol. 1999 April ;154(4):981-6; Bubendorf et al. Cancer Res. Feb. 15, 1999 (4):803-6 and Bubendorf et al. J Natl Cancer Inst. Oct. 20, 1999 ;91(20):1758-64). This new technology has already proven useful for rapidly characterizing the prevalence and prognostic significance of differentially expressed genes identified using cDNA array technology (see, e.g. Bubendorf et al. J Natl Cancer Inst. Oct. 20, 1999;91(20):1758-64; Moch et al. Verh Dtsch Ges Pathol. 1999;83:225-32. German and Barlund et al. J Natl Cancer Inst. Aug. 20, 2000 ;92(15):1252-9) as well as genes involved in cancer development and progression (see, e.g. Bubendorf et al. Cancer Res. Feb. 15, 1999;59(4):803-6 and Bubendorf et al. J Natl Cancer Inst. Oct. 20, 1999;91(20):1758-64). Tissue microarrays have also been useful in identifying genes that are targets of chromosomal amplification (see, e.g. Barlund et al. Cancer Res. Oct. 1, 2000; 60(19):5340-4 and Richter et al. Am J Pathol. 2000 September ;157(3):787-94) as well as to study the expression patterns of putative tumor suppressor genes (see, e.g. Bowen et al. Cancer Res. Nov. 1, 2000;60(21):6111-5).
A variety of technical problems exist with the current methodology, however, relating to the fact that the arrayed samples have to be pre-fixed and embedded in paraffin. The quality of the studies performed on sections from tissue array technology may be limited by the fixation methods used on the original sample. Buffered formalin solutions (and related compounds) are among the most widely used tissue fixatives. These chemicals fix the tissue by acting as progressive cross linkers between proteins and nucleic acids, by introducing modifications in RNA (adding mono-methyl groups to its bases), and by producing coordinate bonds for calcium ions; these processes can damage RNA and alter target antigenic structure by blocking or damaging antibody binding sites (see, e.g. Masuda et al. Nucleic Acids Res. Nov. 15, 1999;27(22):4436-43 and Werner et al. Am J Surg Pathol. 2000 July;24(7):1016-9. Review). Formalin fixation-induced alterations can make in-situ analysis of DNA, RNA, and proteins suboptimal and variations in the duration of fixation can effect the quality and reproducibility of results (see, e.g. Kononen et al. Nat Med. 1998 July;4(7):844-7; Werner et al. Am J Surg Pathol. 2000 July;24(7):1016-9, Review and Specht et al. Am J Pathol. 2001 February;158(2):419-429). Artisans attempt to overcome fixation problems for FISH by uniformly pre-fixing tissues in cold ethanol and embedding in paraffin (see, e.g. Kononen et al. Nat Med. 1998 July;4(7):844-7), but this approach is not optimal for array analysis of some proteins or for RNA using in situ hybridization. Paraffin embedding of ethanol-fixed tissue does not prevent RNA degradation (see, e.g. Goldsworthy et al. Mol Carcinog. 1999 June;25(2):86-91). In addition, while ethanol fixation of tissue and subsequent paraffin embedding circumvents formalin fixation-related problems introduced by crosslinking, there are still problems relating to the embedding, and/or deparaffinization processes such as temperature-induced antigenic alterations introduced during the embedding process (see, e.g. Werner et al. Am J Surg Pathol. 2000 July;24(7):1016-9, Review; Battifora et al. J Histochem Cytochem. 1986 August;34(8):1095-100 and Penault-Llorca et al. J Pathol. 1994 May;173(1):65-75).
Consequently there is a need in the art to identify additional methods that allow for the optimal preservation of biological molecules such as polypeptides and polynucleotides to be analyzed in such arrays. The present invention meets this need in the art by providing methods that circumvent problems associated with traditional paraffin arrays.
The invention disclosed herein improves upon existing tissue microarray technology by arraying the specimens into a recipient block comprising tissue embedding compound. Tissue is not fixed prior to embedding, and sections from the array are evaluated without fixation or post-fixed according to the appropriate methodology used to analyze a specific gene at the DNA, RNA, and/or protein levels.
The invention disclosed herein includes a number of embodiments. A typical embodiment of the invention is method of preparing a tissue microarray by embedding a non-fixed biological sample in the tissue microarray block, wherein the tissue microarray block comprises frozen tissue embedding compound. A related embodiment of the invention includes a method of preparing a tissue microarray comprising the steps of: preparing a tissue microarray block for receipt of a biological sample, wherein the tissue microarray block comprises frozen tissue embedding compound; removing a core sample of a biological sample from a frozen donor block comprising tissue embedding compound; and then placing the core sample of the biological sample into an array within the tissue microarray block. Yet another embodiment of the invention is a method of preparing a biological sample for microarray analysis comprising the steps of: preparing a tissue microarray block for receipt of a biological sample; freezing the biological sample in tissue embedding compound; removing a core sample of the biological sample from the frozen tissue embedding compound; and then placing the core sample of the biological sample into an array within the tissue microarray block, wherein the tissue microarray block comprises frozen tissue embedding compound.
A number of variations on these methods are disclosed herein. In preferred embodiments for example, the biological sample is prepared for placement into the tissue microarray block by removing a core sample of frozen biological material from a donor block comprising frozen tissue embedding compound. Preferably, the biological material is removed from the frozen tissue embedding compound with a coring means having a temperature of less than about 4 degrees centigrade. In yet another embodiment of the invention, a slice of an about 4 xcexcm section of the frozen tissue microarray block comprising a portion of the biological sample is removed for subsequent analysis. In such embodiments of the invention, the tissue can be fixed after being embedded in the frozen tissue embedding compound.
Another embodiment of the invention includes a process for preparing a biological sample for microarray analysis comprising embedding a non-fixed biological sample into an array within a block comprising frozen tissue embedding compound. A closely related embodiment includes a biological sample for microarray analysis prepared by this process. Embodiments of the invention also include compositions comprising an array of biological samples having at least one non-fixed biological sample embedded in a tissue microarray block, wherein the tissue microarray block comprises frozen tissue embedding compound.
The invention also provides article of manufacture or kit comprising one or more polypeptide and/or polynucleotide probes and a tissue embedding medium.
Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications.